1. Field of the Invention
The present invention relates to isolation, identification, synthesis, expression and purification of antibodies reactive with factor IX (FIX)/factor IXa (FIXa) and especially the FIX/FIXa Gla domain. In particular aspects, the invention provides human antibodies reactive with the human FIX/FIXa Gla domain. The invention further relates to compositions, especially pharmaceutical compositions, articles of manufacture, and methods of inhibiting the activation of FIX/FIXa and inhibiting FIX/FIXa dependent coagulation.
2. Description of Related Disclosures
Factor IXa is a vitamin K dependent plasma serine protease that participates in both the intrinsic and extrinsic pathways of blood coagulation. The NH2 terminal 43 amino acids (Gla domain) of factor IXa and its zymogen factor IX contain 12 Gla residues formed by the vitamin K-dependent carboxylation of Glu residues. The Gla domain is followed by two epidermal growth factor (EGF) type domains, followed by a carboxy terminal serine protease domain.
The Gla domain of FIX/FIXa contains important structural determinants for interaction with high affinity binding sites on vascular endothelial cells and platelets (Heimark et al., (1983) Biochem. Biophys. Res. Commun. 111:723-731; Ahmad et al., (1994) Biochem. 33:12048-12055; Ryan et al., (1989) J. Biol. Chem. 264:20283-20287; Toomey et al., (1992) Biochemistry 31:1806-1808; Cheung et al., (1992) J. Biol. Chem. 267:20529-20531; Rawala-Sheikh et al., (1992) Blood 79:398-405; Cheung et al., (1996) Proc. Natl. Acad. Sci. USA 93:11068-11073; Prorok et al., (1996) Int. J. Pept. Prot. Res. 48:281-285; Ahmad et al., (1998) Biochemistry 37:1671-1679). In the presence of Ca++ and Mg++ the FIX/FIXa Gla domain adopts different conformations. Coagulation reactions, such as FIX/FIXa-mediated activation of FX proceed with high efficiency on the surface of activated platelets (Ahmad and Walsh (1994) Trends Cardiovasc. Med., 4:271-277).
Antibodies that bind the FIX/FIXa Gla domain have been shown to inhibit FIX/FIXa function, such as cell binding (Cheung et al., (1996) supra; clotting activity (Sugo et al., (1990) Thromb. Res. 58:603-614) and FIX/FIXa activation by FXI (Sugo et al., (1990) supra; Liebman et al., (1987) J. Bio. Chem. 262:7605-7612). Rabbit and murine antibodies to FIX/FIXa have been shown to bind to the C- and N-terminal region of the Gla domain (Liebman et al., (1993) Eur. J. Biol. Chem. 212:339-345 and Sugo et al., (1990) Thromb. Res. 58:603-614). Antibodies reactive with human FIX/IXa have been shown to inhibit the activation of FIX to FIXa and inhibit coagulation in a FIXa dependent assay (Blackburn et al., (1997) Blood 90: Suppl. 1:424-425a). Active site inhibited FIXa attenuates thrombosis in vivo (Wong et al., (1997) Thromb. Haemost. 77:1143-1147; Benedict et al., (1991) J. Clin. Invest. 88:1760-1765; Spanier et al., (1998) J. Thoracic Cardiovasc. Surgery 115:1179-1188).
The present invention provides isolated antibodies, antibody fragments, especially human antibodies and antibody fragments, reactive with the factor IX or factor IXa Gla domain. In preferred aspects the antibodies or antibody fragments inhibit an activity associated with blood coagulation factor IX or IXa. Advantageously, the antibodies of the present invention provide for the preparation of potent pharmaceutical compositions comprising the antibodies. The pharmaceutical compositions provide for low dose pharmaceutical formulations for the treatment of acute and chronic thrombotic disorders without compromising normal hemostasis.
In one embodiment, the invention provides an antibody or antibody fragment that reacts with human factor FIX/FIXa and especially the human FIX/FIXa Gla domain. Representative antibody fragments include Fv, scFv, Fab, F(abxe2x80x2)2 fragments, as well as diabodies and linear antibodies. These fragments may be fused to other sequences including, for example, a xe2x80x9cleucine zipperxe2x80x9d or other sequence and include pegylated sequences or Fc variants used to improve or modulate half-life. Representative antibodies or antibody fragments comprise three complementarity-determining regions (CDRs) referred to herein as CDR1, CDR2 and CDR3. The amino acid sequences of the CDR polypeptides are selected from those of the exemplary antibody fragments 10C12, 11C5, 11G9, 13D13H6 and 14H9 and variants thereof. Preferred antibodies are selected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein the CDRs of Ab1-Ab6 correspond to those of 10C12, 11C5, 11G9, 13D1, 13H6 and 14H9, respectively.
In one embodiment, the composition of the present invention is an antibody polypeptide and the invention encompasses a composition of matter comprising an isolated nucleic acid, preferably DNA, encoding the polypeptide of the invention. According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell comprising the vector, as well as a host cell comprising the vector.
The present invention further extends to therapeutic applications for the antibody compositions described herein. Thus the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an antibody or antibody fragment of the invention. The invention includes kits and articles of manufacture comprising the antibody compositions of the invention. Kits and articles of manufacture preferably include:
(a) a container;
(b) a label on said container; and
(C) a composition comprising an antibody or antibody fragment of the invention contained within said container; wherein the composition is effective for treating a coagulation disorder and the optional label on said container indicates that the composition can be used for treating a coagulopathic disorder. The kits optionally include accessory components such as a second container comprising a pharmaceutically-acceptable buffer and instructions for using the composition to treat a coagulation related disorder.
Pharmaceutical compositions comprising the antibodies or antibody fragments can be used in the treatment or prophylaxis of thrombotic or coagulopathic diseases or disorders and include, for example, methods of treating a mammal for which inhibiting a FIX/FIXa mediated event is indicated. The methods comprise administering a therapeutically effective amount of a pharmaceutical composition of the invention to the mammal. Such indications include, deep venous thrombosis, arterial thrombosis, unstable angina, post myocardial infarction, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transluminal coronary angioplasty (PTCA), stroke, tumor growth, invasion or metastasis, inflammation, septic shock, hypotension, ARDS, atrial fibrillation and DIC. The compositions of the present invention may also be used as an adjunct in thrombolytic therapy.